The long-term goal of this application is to reconstitute the cardiac pacemaker channels in vitro and apply the HCN pacemaker channel proteins to the gene therapy of arrhythmias. Based on recent advances in HCN cloning, mRNA distribution, functional expression of channels in different cell types, and regulation by beta subunit and tyrosine phosphorylation (TYR PHOS), this application attempts to assess the individual contribution of three cardiac HCN isoforms to the distinct properties of the pacemaker current i(f) and examine the mechanism and cellular function of tyrosine kinases related to pacemaker activity through i(f). Employing a combined electrophysiology (two-electrode voltage clamp, single-cell perforated patch clamp) and molecular biology (RNAi, mutagenesis, recombinant DNA) techniques, we will investigate 1) the effects of tyrosine kinase activation on i(f) and HCN channel properties; 2) the role of a nonreceptor tyrosine kinase, Src, in the modulation by both receptor/nonreceptor tyrosine kinases of pacemaker channels; 3) the molecular mechanism of how TYR PHOS differentially regulates three cardiac HCN isoforms; and 4) the contribution of each HCN isoform and TYR PHOS on HCN channels to the pacemaker activities in different heart regions (e.g., SA node and ventricle). Xenopus laevis oocytes, human embryonic kidney 293 (HEK293) cells, and rat neonatal ventricular myocytes will be used to provide a quick assessment on HCN modulation by TYR PHOS, a mammalian background for regulatory studies of HCN channels, and the physiological significance of the role HCN isoforms may play in the TYR PHOS of i(f) in vivo. Sinus node disease and atrial fibrillation are among the most common arrhythmias in patients. I(f) is an important contributor to the regulation of cardiac pacemaker activity. Understanding the modulation of HCN channels that encode i(f) should have significant diagnostic and therapeutic implications.